BACTERIA RECOVERED FROM AQUACULTURE IN OMAN, WITH EMPHASIS ON AEROMONAS SPP. THESIS SUMITTED TO THE UNIVERSITY OF STIRLING FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY ALYA SALIM AL-GHABSHI APRIL 2015 INSTITUTE OF AQUACULTURE DECLARATION I hereby declare that the work and results presented in this thesis were conducted by me at the Institute of Aquaculture, University of Stirling, Scotland. The work presented in this thesis has not been previously submitted for any other degree or qualification. The litera- ture consulted has been cited and where appropriate, collaborative assistance has been acknowledged. Signature: ________________________________________ Signature of Principal Supervisor: ________________________________________ Signature of Supervisor: ________________________________________ Date: ________________________________________ I In loving memory of my mother To my husband Hamood Al-Ghabshi and My lovely sons Yousuf and Mohammed II Acknowledgements ACKNOWLEDGEMENTS First of all, I would like to thank the almighty Allah for helping me to finish my study successfully. I would like to express my warmest gratitude to my supervisor’s and mentors Professor Brian Austin and Dr. Mags Crumlish, who has introduced me to the world of science. Their support and guidance gave me to find out the way to continue my research in the field and laboratory. During writing up this thesis, their valuable comments, suggestions, excellent proofreading skills and encouragement helped me to complete this work with the incredible optimism. I am also extremely grateful for the assistance and guidance from Dr. Ponnerassery S. Sudheesh and from staff members at the Institute of Aquaculture (stirling) particularly, Mrs. Debbie Faichney, Mrs. Jan Seggie, Dr. John Taggart, Mrs. Jacquie Ireland, Mr. Niall Auchinachie, Dr. Amaya Albalat, Professor Randolph Richards. Without their training, perseverance and patience I would never have learned the techniques required to finish this project. I am also thankfull Amal Al- Maashari , Umm kulthum Al Kindi, Nasser Al-Aboudi, Wa- heeda Al-Amri, Mr. Denny Conway, Mochamad Syaifudin , Greta Carmona-Antonanzas , Gil Ha Yoon and Dr. Andy Shinn for their kind help in my research. Special thanks to my dear friends Olga Rubio Mejia, Taslima Khanam and to all my good friends in Stirling who helped me to find a home abroad. My special thanks to my sisters, nieces and nephews for they endless love that encour- aged me a lot. I’m deeply grateful to my husband Hamood Al-Ghabshi, for his endless III Acknowledgements love and support that helped me to keep going, especially during the time I was very much frustrated. I also wish to thank my lovely sons Yousuf and Mohammed for doing their best to under- stand their mother who had to be confined to her study for such a long time. IV Abstract ABSTRACT Aquaculture is being seriously considered as a promising sustainable industry in the Sul- tanate of Oman. Fish farming commenced in Oman in 1986, but it was only in 2011 that it became a more commercially driven sector. While worldwide aquaculture production is expected to rise to meet the shortage in capture fisheries, there is a parallel requirement to identify potential threats to the health and welfare of existing aquatic farmed stocks and to take appropriate steps to mitigate them. As aquaculture in Oman is in an early stage of development, it is important to acquire baseline data on the existence and prevalence of aquatic diseases and pathogens to help the Government make policy decisions to develop health management regimes applicable for Omani aquaculture. Therefore, this study was conducted to evaluate current farming practices of tilapia in Oman, to investigate the bacterial species composition and distribu- tion from different sites in some of the economically important fish species, and to study the characteristics and pathogenicity of Aeromonas species. The current practices were studied for 9 Nile tilapia (Oreochromis niloticus) farms from four areas (Al Batinah, Ad Dhahirah, Ad Dakhiliyah and Ash Sharqiyah North) during the period of September to November 2012 by using questionnaires and interviews with the farm owners and staff. In total 417 fish representing 5 target species were chosen on the basis of the commercial importance and their potential for aquaculture in Oman, including red spot emperor (Le- V Abstract thrinus lentjan), king soldier bream (Argyrops spinifer), white spotted rabbit fish (Siganus canaliculatus), abalone (Haliotis mariae) and tilapia (Oreochromis niloticus). The fish were collected from 5 main sampling areas in Oman (Muscat, Mudhaibi, Manah, Sohar and Salalah) based on the Atlas of suitable sites for aquaculture in Oman to investigate the bacterial species composition and distribution. The animals were examined for clini- cal signs of disease prior to routine bacteriology. Bacterial isolates were recovered using traditional methods and identified to species level using phenotypic and molecular ap- proaches using 16S rDNA, 16S rDNA RFLP and 16S rDNA sequencing. Experimental fish challenge studies were also conducted using both live bacterial cells and ECP protein to investigate the pathogenicity of Aeromonas isolates. In addition, the presence of some virulence factors was investigated using both phenotypic and genotypic methods. The results of this study showed that, the most farms in the Oman follow very similar farming practices. The major proportion of the tilapia is consumed within the local com- munities. A number of farmers have experienced mortalities, which were considered to be attributable to poor water quality, overcrowding or due to excessive feeding. Farmers fac- ing fish mortalities tended not to record the problems due to a lack of understanding of the concept of fish farm management. There is a regulation about aquaculture and related quality control, but it has not yet been implemented in an appropriate manner in Oman. From the diverse group of bacteria recovered from wild and farmed fish, 83% of the total isolates comprised Gram negative, rod-shaped bacteria. The most frequently isolated groups from marine and cultured fish were Aeromonas spp., Vibrio spp., Sphingobacte- rium spp., Micrococcus spp. and Staphylococcus spp., with Aeromonas spp. being the VI Abstract predominant group representing 25% of the isolates recovered in this study. Identification of the Aeromonas spp. showed 57% agreement between the results of phenotypic and genotypic methodologies, and determined 6 species as the dominant organisms, i.e. A. veronii, A. jandaei, A. caviae, A. trota, A. encheleia and A. salmonicida. 65% of the iso- lates shared 99% 16S rDNA sequence similarity with the closest sequences in GenBank, and the dominant species was A. veronii. In conclusion, the Aeromonas isolates recovered from fish with clinical signs of disease showed heterogeneity in their identification profiles and their pathogenicity. VII List of attendance conference and published articles LIST OF ATTENDANCE CONFERENCE AND PUBLISHED ARTICLES Presentation Alya AL-Ghabshi, Brian Austin and Mags Crumlish (18-2-2015). Aeromonas spe- cies diversity in the aquatic environment of Oman. PhD Research Conference on sustainable aquaculture 2015 (Stirling University). Article Alya AL-Ghabshi. (March 2015). Disease data in young industry. fish farmer, www.fishamer-magazine.com ,38,P.49 VIII Table of contents TABLE OF CONTENTS DECLARATION ................................................................................................................ I ACKNOWLEDGEMENTS ............................................................................................III ABSTRACT ...................................................................................................................... V LIST OF ATTENDANCE CONFERENCE AND PUBLISHED ARTICLES ....... VIII TABLE OF CONTENTS ............................................................................................. VIII LIST OF FIGURES ...................................................................................................... XVI chapter 1 . General introduction ...................................................................................... 1 1.1 Background ................................................................................................................ 1 1.2 Fish and fisheries in Oman ......................................................................................... 2 1.3 Fish pathogens ........................................................................................................... 5 1.4 Aeromonas ................................................................................................................. 7 1.4.1 Disease distribution and clinical signs associated with Aeromonas infections ....... 9 1.4.2 Clinical signs of disease .......................................................................................... 10 1.4.3 Geographical distribution ....................................................................................... 13 1.4.4 Diagnostic methods for the detection and characterization of Aeromonas spp. .. 13 1.4.5 Pathogenicity and virulence factors ....................................................................... 16 1.5 Research objectives ................................................................................................. 26 1.6 References................................................................................................................ 27 chapter 2 . Current